Two successful embryo transfers of mini-donkey embryos in Brazilian Northeastern jennies using an alternative method: Case report

Pregnancy rates after embryo transfer (ET) are disappointing in donkey species. This study aims to report two successful ET of mini-donkey embryos using Brazilian Northeastern jennies as recipients. Eighteen embryo flushes were performed 9 days post-ovulation in two non-pregnant mini-donkeys jennies (11 and 7 cycles per jenny). Eleven embryos (61%, 11/18) were collected and transferred to Brazilian Northeastern jennies 4–6 days post-ovulation by conventional (n = 6) or an alternative (n = 5) technique. The alternative method consisted of inserting a Polansky equine vaginal speculum smeared with lubricant in the vagina of the recipient jenny. The arms of the speculum were extended to allow the visualization of the cervix. Then, using an adapted crafted, elongated, toothed tissue grasping forceps, the external cervical os was held, and the cervix was gently pulled backward, aiming to straight the cervical canal. The ET gun was inserted through the vagina and cervix by visual inspection, and the embryo was released into the uterine lumen. All embryos collected were Grade 1 and classified as Expanded Blastocysts. No jennies become pregnant after conventional ET (0/6), whereas two recipient jennies (40%, 2/5) become pregnant and delivered offspring in the following year after ET using the alternative technique. In conclusion, Brazilian Northeastern jennies can be used as embryo recipients using the alternative method proposed in the present study. However, further investigations are needed to improve the knowledge and results of ET in donkey species.     

Assessing spillover from marine protected areas and its drivers: A meta‐analytical approach

Overfishing may seriously impact fish populations and ecosystems. Marine protected areas (MPAs) are key tools for biodiversity conservation and fisheries management, yet the fisheries benefits remain debateable. Many MPAs include a fully protected area (FPA), restricting all activities, within a partially protected area (PPA) where potentially sustainable activities are permitted. An effective tool for biodiversity conservation, FPAs, can sustain local fisheries via spillover, that is the outward export of individuals from FPAs. Spillover refers to both: “ecological spillover”: outward net emigration of juveniles, subadults and/or adults from the FPA; and “fishery spillover”: the fraction of ecological spillover that directly benefits fishery yields and revenues through fishable biomass. Yet, how common is spillover remains controversial. We present a meta‐analysis of a unique global database covering 23 FPAs worldwide, using published literature and purposely collected field data, to assess the capacity of FPAs to export biomass and whether this response was mediated by specific FPA features (e.g. size, age) or species characteristics (e.g. mobility, economic value). Results show fish biomass and abundance outside FPAs was higher: (a) in locations close to FPA borders (<200 m) than further away (>200 m); (b) for species with a high commercial value; and (c) in the presence of PPA surrounding the FPA. Spillover was slightly higher in FPAs that were larger and older and for more mobile species. Based on the broadest data set compiled to date on marine species ecological spillover beyond FPAs' borders, our work highlights elements that could guide strategies to enhance local fishery management using MPAs.     

Putative virulence-related genes in Vibrio anguillarum identified by random genome sequencing

The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes.     

Nutrients removed by Kappaphycus alvarezii (Rhodophyta, Solieriaceae) in integrated cultivation with fishes in re-circulating water

The potential of the red alga Kappaphycus alvarezii to remove nutrients was tested to treat effluents of Trachinotus carolinus fish cultivation, and the production of carrageenan in this condition was analyzed. Experiments were conducted in four tanks of 8000 L with approximately 1200 fishes of 30 g each integrated with three tanks of 100 L with 700 g of K. alvarezii, as initial biomass per tank. Seawater was re-circulated between tanks with seaweed and with fish. As a control, three tanks with seawater circulating in an open system were utilized. Seawater samples were collected daily for 10 days and concentrations of nitrate, nitrite, ammonium and phosphate were determined in the inflow and outflow water of the tanks. Significant differences between both collecting points were considered as nutrient removal by the seaweed. Growth rates and carrageenan yields were also analyzed in seaweed cultivated in seawater and in effluents. Growth rates of seaweed cultivated in tanks were lower than those obtained in open sea and in laboratory cultivation. Effluents had concentrations of nitrate and nitrite ca. 100 times higher than in the control. Maximum values of nutrient removal on effluents were: nitrate = 18.2%; nitrite = 50.8%; ammonium = 70.5% and phosphate = 26.8%. All plants survived throughout the experimental period, but some developed “ice–ice”, a disease associated with physiological stress. After the experimental period, some plants selected and cultivated in open sea presented higher growth rates in 40 days, indicating nutrient storage. No significant differences between carrageenan yields of K. alvarezii cultivated in seawater and in the effluents were observed. Our results show that K. alvarezii can be utilized as a biofilter for fish cultivation effluents, reducing the eutrophication process and can also be processed for carrageenan production, which provides an additional benefit to the fisheries.     

Growth,absorption and assimilation efficiency by mature cuttlefish (<Emphasis Type="Italic">Sepia officinalis</Emphasis>) fed with alternative and artificial diets

The effects of feeding three natural frozen diets, grass shrimp (Palaemonetes sp.), crayfish (Procambarus clarkii) and fish (Sardina pilchardus) and two semi-humid artificial diets (based on fish powder) to mature cuttlefish, Sepia officinalis, were analysed. Growth and feeding rates (GR and FR, % BW day⁻¹), food conversions (FC, %), and total protein and lipid composition of the diets were determined. Digestive gland to body weight ratio and absorption efficiency were calculated for each diet. Cuttlefish fed shrimp and crayfish grew larger (1.5 and 1.1% BW day⁻¹, respectively) compared to the other diets. Shrimp promoted the highest FC, followed by crayfish, and sardine. The highest FR was obtained for cuttlefish fed crayfish (8.4% BW day⁻¹). Although both artificial diets were accepted, none produced growth. A positive correlation (r = 0.96) between cuttlefish ingestion rate and digestive gland weight was obtained. Some cannibalism occurred among cuttlefish fed the artificial diets during the last week of the experiment. According to the results obtained, P. clarkii could be used as an alternative prey to shrimp for rearing adult mature (>50 g) S. officinalis.     

Short-term changes in nitrogen availability, gas fluxes (CO2, NO, N2O) and microbial biomass after tillage during pasture re-establishment in Rondônia, Brazil

Anthropogenic conversion of primary forest to pasture for cattle production is still frequent in the Amazon Basin. Practices adopted by ranchers to restore productivity to degraded pasture have the potential to alter soil N availability and N gas losses from soils. We examined short-term (35 days) effects of tillage prior to pasture re-establishment on soil N availability, CO₂, NO and N₂O fluxes and microbial biomass C and N under degraded pasture at Nova Vida ranch, Rondônia, Brazilian Amazon. We collected soil samples and measured gas fluxes in tilled and control (non tilled pasture) 12 times at equally spaced intervals during October 2001 to quantify the effect of tillage. Maximum soil NH₄⁺ and NO₃⁻ pools were 13.2 and 6.3 kg N ha⁻¹ respectively after tillage compared to 0.24 and 6.3 kg N ha⁻¹ in the control. Carbon dioxide flux ranged from 118 to 181 mg C–CO₂ m² h⁻¹ in the control (non-tilled) and from 110 to 235 mg C–CO₂ m² h⁻¹ when tilled. Microbial biomass C varied from 365 to 461 μg g⁻¹ in the control and from 248 to 535 μg g⁻¹ when tilled. The values for N₂O fluxes ranged from 1.22 to 96.9 μg N m⁻² h⁻¹ in the tilled plots with a maximum 3 days after the second tilling. Variability in NO flux in the control and when tilled was consistent with previous measures of NO emissions from pasture at Nova Vida. When tilled, the NO/N₂O ratio remained <1 after the first tilling suggesting that denitrification dominated N cycling. The effects of tilling on microbial parameters were less clear, except for a decrease in qCO₂ and an increase in microbial biomass C/N immediately after tilling. Our results suggest that restoration of degraded pastures with tillage will lead to less C matter, at least initially. Further long-term research is needed.     

Fusarium moniliforme secretes four endopolygalacturonases derived from a single gene product

Extracellular endopolygalacturonase, purified from the pathogenic fungus Fusarium moniliforme, consists of four molecular forms (38, 41·5, 45, and 48·5 kDa, respectively. Three forms (38, 41·5, and 45 kDa) were purified to homogencity by FPLC on a Mono S column followed by electroelution after SDS-PAGE. The N-terminal amino acid sequences of each of the three forms, and of a mixture containing all four forms were shown to be identical to that predicted from the nucleotide sequence of the endopolygalacturonase gene previously cloned from F. moniliforme. Enzymatic deglycosylation experiments revealed the presence of N-linked, high mannose oligosaccharide side-chains on all four forms of endo polygalacturonase. Hydrogen fluoride catalysed chemical deglycosylation of the polygalacturonase mixture yielded a single polypeptide with an apparent molecular mass of 36·2 kDa. Southern blot analysis, carried out at high stringency with an endopolygalacturonase-specific probe on genomic DNA digested with three different restriction enzymes, showed a single hybridizing restriction fragment in all three digests. A single 2·0 Mb chromosome hybridized with the endo polygalacturonase-specific probe, as shown by Southern blot analysis of F. moniliforme chromosomes separated by CHEF electrophoresis. Northern blot analysis revealed only one mRNA species 1350 nt encoding endo polygalacturonase. These data indicate that a single gene encodes the endopolygalacturonases of F. moniliforme.     

A morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with Mycoplasma hyopneumoniae

Porcine enzootic pneumonia (PEN), caused by Mycoplasma hyopneumoniae (Mh), has been described in pigs in all geographic areas. The disease is characterized by high morbidity and low mortality rates in intensive swine production systems. A morphologic and immunohistochemical study was done to determine the cellular populations present in lung parenchyma of infected pigs, with special attention to the bronchus-associated lymphoid tissue (BALT). Polyclonal and monoclonal antibodies were used for the detection of antigens of Mh, T lymphocytes (CD3+, CD4+, and CD8+), IgG+ or IgA+ lymphocytes, and cells containing lysozyme, S-100 protein, major histocompatibility complex class II antigen or myeloid-histiocyte antigen. Findings in lung tissues associated with Mh infection were catarrhal bronchointerstitial pneumonia, with infiltration of inflammatory cells in the lamina propria of bronchi and bronchioles and alveolar septa. Hyperplasia of mononuclear cells in the BALT areas was the most significant histologic change. The BALT showed a high morphologic and cellular organization. Macrophages and B lymphocytes were the main cellular components of germinal centers. T lymphocytes were primarily located in perifollicular areas of the BALT, lamina propria and within the airway epithelium, and plasma cells containing IgG or IgA at the periphery of the BALT, in the lamina propria of bronchi and bronchioles, in alveolar septa, and around bronchial submucosal glands. The hyperplastic BALT in PEN cases consisted of macrophages, dendritic cells, T and B lymphocytes, and IgG+ and IgA+ plasma cells. CD4+ cells predominated over CD8+ cells. Local humoral immunity appears to play an important role in the infection.     

Postsurgical intra-incisional 5-fluorouracil in dogs with incompletely resected, extremity malignant spindle cell tumours: a pilot study

The safety and efficacy of intra‐incisional 5‐fluorouracil (5‐FU) in the management of incompletely resected malignant spindle cell tumours of extremities was evaluated in six dogs. After marginal surgery, the dogs underwent weekly intra‐incisional 5‐FU for a minimum of six cycles. Treatment was well tolerated by all dogs, with no systemic adverse effects and only one episode of local cutaneous hyperpigmentation, which completely and spontaneously resolved. Median follow‐up for all the dogs was 546 days (mean 619; range 297–1207). At the date of analysis, four dogs were still alive with no evidence of local recurrence, and two dogs had died as a result of their disease. The cause of death was development of distant metastases in one dog and tumour regrowth in the other. Despite the small sample size, this study documents that intra‐incisional 5‐FU chemotherapy is a safe and efficacious adjuvant treatment in the case of incompletely resected malignant spindle cell tumours in dogs and that long disease control can be achieved.